Neuroblastoma-associated regulator gene

ABSTRACT

PCT No. PCT/EP92/02962 Sec. 371 Date Jun. 23, 1994 Sec. 102(e) Date Jun. 23, 1994 PCT Filed Dec. 19, 1992 PCT Pub. No. WO93/13205 PCT Pub. Date Jul. 8, 1993Described is a gene situated in the region of the neuroblastoma consensus deletion 1p36.2-p36.1 and which codes for a helix-loop-helix protein with the designation HEIR-1. The gene is affected significantly by allelic tumor deletions in neuroblastomas and correlates inversely both N-myc overexpression in tumors and with N-myc expression in normal development. The cDNA and antibodies coding for HEIR-1 are used for the diagnosis of pathological conditions associated with aberration in the region of the neuroblastoma consensus deletion.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a gene which is affected significantly bytumour-specific deletions in human neuroblastomas and is involved intumour genesis.

2. Description of Related Art

A group of proteins which have been shown to act predominantly asactivators of transcription share common motifs for dimerisation and DNAbinding (Jones, 1990). The dimerisation domain is an amphipathicHelix-Loop-Helix (HLH)-region, and the DNA-binding is made easier by asection of basic amino acids which precede the HLH domain (Murre et al.,1989a,b). Proteins which contain this basic Helix-bop-Helix pattern(bHLH) may form homo- and heterodimers. The modulation of thetranscriptional activation by bHLH factors is brought about by anothergroup of dimerising proteins. This was established in the course ofidentifying the Id protein, a protein which also contains aHelix-Loop-Helix pattern but which lacks the basic region (HLH proteins)(Benezra et a., 1990). Id forms heterodimers with some members of thefamily of the bHLH transcription factors. Since it lacks the basicregion which is responsible for DNA binding, heterodimers which containId cannot bind DNA. Therefore, bHLH proteins are most probably regulatednegatively by HLH proteins such as Id. Other examples of regulatory bHLHand HLH protein interactions are the genes of the Drosophilaachaete-scute-complex and the extramacrochaetae (emc) gene (Ellis etal., 1990; Garell and Modolell, 1990). These genes have a function indeveloping the sense organs of the peripheral nervous system (Ghysen andDambly-Chaudiere, 1989). In general, genes which code forHelix-Loop-Helix proteins are presumed to be involved in controllingcell differentiation.

Cell differentiation is one of the processes which is affected byneoplastic transformation. Therefore, the function of genes involved inthe these processes must be disrupted in tumour cells. The appearance ofboth dominant and recessive mutations in tumours must reflect the normalfunction of the affected genes as either positive or negativeregulators. Up till now, Helix-Loop-Helix proteins have only been foundto have effects based on dominant mutations, eg. the oncogenicactivation of myc genes (Luscher and Eisenmann, 1990; Zimmerman and Alt,1990). However, in view of the different functions of the genes codingfor HLH proteins it could also be borne in mind that genes of this typeare affected by recessive mutations and therefore have properties whichcorrespond to the tumour suppressor genes.

Allelic deletions in specific regions of the genome, which occursignificantly frequently in tumour genomes, are used as marking pointsfor the position of tumour suppressor genes (Weinberg, 1991). Ininvestigations of human neuroblastomas a consensus deletion was definedin chromosome lp36.2-p36.1 (Weith et al., 1989). The allelic loss ofthis section in about 80-90% of the tumours investigated led to thesupposition that a gene which prevents tumours is located in thisregion.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The aim of the present invention was to isolate such a gene.

The following procedure was used to solve the problem: In order toidentify genes in the human chromosome lp36 region, a panel oflp36-specific microcloned DNA probes (Martinsson et al., 1989) was usedto identify CpG Islands. CpG Islands represent DNA sections which arerich in CpG and 0.5-1 kbp long, which are associated with the 5' ends ofnumerous genes (Bird, 1986). The cytosine groups in the Islands are notmethylated and therefore corresponding sequences ofmethylation-sensitive, rarely cutting restriction enzymes arerecognised. Consequently, individual probes were hybridised with pulsefield electrophoresis blots of genomic DNA which had been subjected toeither single or double digestion with rarely cutting restrictionenzymes. Those DNA probes which were located between two CpG Islandswere expected to hybridise with fragments of the same size in differentsingle and double digestion. The probe designated p1-112B, mapped in1pter-p36.12, showed a band of 25 kb in Bss HII, Eag I, Nae I, Nar I,Sac II and Sma I digestion (FIG. 1A). The band removed the same size inDNA which had been digested with combinations of these enzymes.Therefore, p1-112B is obviously located in a 25 kb section between twoclusters of recognition sites for rarely cutting restriction enzymes(FIG. 1B).

In order to clone these clusters a genomic cosmid library was searchedwith p1-112B and a positive clone was isolated. The cosmid, which wasdesignated C1-112B, contained a 37 kb genomic DNA insert. Restrictionmapping of this clone with rarely cutting restriction enzymes yieldedtwo clusters of restriction cutting sites roughly 25 kb away from eachother (FIG. 2). One of the clusters was found to be located in thep1-112B probe. The other cluster was sub-cloned into a 9.0 kb EcoRIfragment (C1-112B/9.ORI). The hybridisation of C1-112B/9.ORI on Southernblots with DNA which had been cut out of the clusters with the enzymesshowed that the rare cutting sites in the genomic DNA are obviously notmethylated and that the cluster of rare cutting sites constitutes a CpGIsland. Similar results were obtained for the cluster contained in thep1-112B probe.

CpG Islands constitute the 5' end of genes and often include part of thefirst exon (Bird, 1986). In order to determine whether C1-112B/9.ORIrecognises a transcript, a northern blot of total RNA from HeLa cellswas investigated using the probe. A positive band was detected at about1.2 kb. Then a HeLa cDNA library was searched using C1-112B/9.ORI. Twodifferent cDNA clones of different lengths were isolated. The two cloneswere sequenced; sequence analysis yielded an identical sequence for bothclones, apart from the fact that 200 bp were missing from the shorterclone at the 5' end. The longer clone was designated HEIR-1 and used forsubsequent experiments. Its sequence (SEQ ID NO: 1) comprises 982nucleotides and contains a single open reading frame, beginning with astart codon at position 37 and ending with a TGA stop codon at position481. In the reading frame with the first ATG, an additional possiblestart codon was found at position 124. The 3'-untranslated regioncontains a poly(A) signal at position 971. An ATTTA motif, which wasshown to be responsible for the short half life of the mRNA (Brawerman,1989) was also found in an AT-rich section of the 3'-untranslated partof the message.

Since no stop codon was found in the reading frame 5' from the firststart codon, it was not ruled out initially that the reading frameextends further in the 5' direction.

Direct sequencing of the region 5' from the ATG codon at position 124 ofthe cosmid C1-112B showed that the sequence of the HEIR-1 cDNA clonediffers from the genomic DNA in this section. Direct sequence analysisof the 5' region (nucleotide 1-123) from the cDNA clone showed nohomology with the genomic locus. It was therefore assumed that this DNAsection of the HEIR-1 cDNA clone is a cloning artifact.

The genomic DNA fragment p1-112B is located on chromosome 1pter-p36.12.In order to confirm the position of the HEIR-1 gene, a series of(HumanXMouse) microcell hybrids (Martinsson et al., 1989) were used formapping by means of Southern blot (Southern Mapping). The hybrid celllines each contain the human chromosome 1 with various deletions of the1p arm as the only human material on a mouse background. The HEIR-1-cDNAwas tested on a Southern blot containing EcoRI-digested DNA of the fourhybrid cell lines (FIG. 6). A positive human fragment was found only inthe DNA of the particular hybrid which contained an intact chromosome 1,but not in any of the hybrids which had deletions in the 1pter-p36.12region. From this, it could be concluded that HEIR-1 is situated in thesame chromosome regions as the genomic probe which was initially used toidentify the CpG Island, and that the gene found is consequently the onewhich was being sought.

The deletion break off point of the hybrid cell line 20-EA3characterises the proximal limit of the neuroblastoma consensus deletion(Martinsson et al., 1989; Weith et al., 1989). Since HEIR-1 is locateddistally of this break off point an investigation was also made to seewhether the gene locus is contained within the limits of the consensusdeletion. For this purpose, a neuroblastoma tumour designated N-29(Weith et al., 1989) was investigated with an allelic deletion thedistal break off point of which marks the distal limit of consensusdeletion. Using a restriction fragment length polymorphism (RFLP) anallelic deletion in the HEIR-1 locus of this tumour was detected. Thisconstituted proof that HEIR-1 is located in the neuroblastoma consensusdeletion.

The present invention relates to a new human DNA which is located in theneuroblastoma consensus deletion 1p36.2-p36.1 and which contains theregion coding for a Helix-Loop-Helix protein designated HEIR-1.

According to one aspect of the invention the DNA is genomic DNA.

According to another aspect of the invention the DNA is a cDNA.

The open reading frame of the HEIR-1 cDNA codes for a protein of 119amino acids which contains a Helix-Loop-Helix motif. The coding regionof the cDNA and the derived polypeptide sequence are shown as SEQ ID NO:3 and SEQ ID NO: 4. (The open reading frame found in the isolated cDNAclone which has the sequence shown in SEQ ID NO: 1, would code for apolypeptide of 148 amino acids; the correspondingly derived sequence isshown in SEQ ID NO: 2.) Sequence comparison with members of the familyof the HLH transcription factors yielded 95.8% identity with the murineHLH 462 (Christy et al., 1991) over all 119 amino acids of HLH 462 (FIG.3B). The differences in amino acids between HEIR-1 and the murine HLH462 protein are obtained by replacing 5 non-polar amino acids with 5polar amino acids. These changes are outside the HLH motif. TheN-terminal 29 amino acids of the protein derived from the cDNA clone arenot contained in the mouse protein (FIG. 3B). It was therefore assumedthat the second ATG at position 124 coincides with the start codon ofHLH 462, i.e. the first ATG is skipped over and the second ATG is thecorrect start codon of HEIR-1. This assumption was confirmed by directsequencing of the genomic locus (see above).

The HLH motif of HEIR-1, like that of HLH 462 (Christy et al., 1991),shows clear homology with the HLH motifs of Id and the Drosophila emcprotein. In particular, 11 of the 16 amino acids of the second Helix areconserved between HEIR-1, Id and emc (FIG. 4). Like Id, emc and HLH 462,HEIR-1 lacks a basic region which is necessary for a specific DNAbinding and is found in bHLH proteins. However, a clear similarity wasalso found with the HLH motifs of bHLH proteins such as MyoD (Davis etal., 1987), c-myc (Bernard et al., 1983) and E47 (Murre et al., 1989a).

The availability of HEIR-1-cDNA presupposes the production of HEIR-1 inlarger quantities as a recombinant protein. Production takes place insuitable prokaryotic or eukaryotic host organisms, e.g., E. coli, yeastor cells of higher organisms. The techniques used to produce recombinantpolypeptides are well known to those skilled in the art; vectors,control sequences and so on which are suitable for the particularexpression host can be found in the relevant text books (e.g. Sambrooket al., 1989, Molecular Cloning a Laboratory Manual. Cold Spring HarborLaboratory).

The invention thus relates, according to a further aspect, torecombinant DNA molecules containing the DNA coding for HEIR-1 as wellas expression control sequences functionally connected therewith, andthe host organisms transformed therewith.

The method of restriction mapping is used to determine whether thecomplete HEIR-1 gene is contained in the isolated cosmid clones. Inorder to do this, cosmid DNA is cut with restriction enzymes,gel-fractionated, transferred to membranes and hybridised with theHEIR-1 cDNA. It is taken as proof of completeness if the cosmid DNA hasall the restriction fragments which are homologous to the cDNA. Othergenomic clones can be obtained from the genomic libraries by screeningwith the cDNA according to the invention. The 5'-regulatory region wasdetermined by sequencing with the aid of a primer which wascomplementary to the 5' region of the coding gene section. Sequencingwas carried out in the genomic DNA clone C1-112B/9.ORI. The sequence,including the start codon, is shown in SEQ ID NO: 5.

The invention further relates to the recombinant HEIR-1 protein.

The availability of the protein makes it possible to prepare anti-HEIR-1antibodies, preferably monoclonal anti-HEIR-1 antibodies.

The present invention thus further relates to antibodies against HEIR-1,preferably monoclonal antibodies, as well as hybridoma cells whichproduce such antibodies, and processes for preparing them.

The preparation of monoclonal antibodies is familiar to those skilled inthe art; suitable methods are described in the relevant text books, eg.Harlow and Lane, 1988). The preparation is based essentially on themethod described by Kohler and Milstein, 1975, in which animals such asmice are immunised with the antigen, B-lymphocytes from the immunisedanimals are fused with myeloma cells and the antibodies are obtainedfrom the resulting hybridomas.

Since the HEIR-1 gene is located in a genomic region which is frequentlyaffected by allelic deletions in neuroblastomas, a number ofneuroblastomas have been searched for deletions of the HEIR-1 locus.Using an Apa I restriction fragment length polymorphism (RFLP) test forthis locus, a so-called LOH (loss of heterozygosity) analysis wascarried out. Southern blots prepared with DNAs from 16 differentneuroblastomas and the DNAs from the corresponding normal tissue,digested with Apa I, were investigated using HEIR-1-cDNA as the probe.

Heterozygosity of the locus was found in the DNA of 11 different normaltissues. Of these 11 cases, 7 exhibited a tumour-specific loss of anallele. Among these 7 tumours there were two which had been shown tohave allelic deletions and which constituted the proximal and distallimits of the neuroblastoma consensus deletion (FIG. 8) (Weith et al.,1989). Since the HEIR-1 probe in both cases showed a loss ofheterozygosity the locus could obviously be put down to consensusdeletion.

In order to detect whether the HEIR-1 gene is transcribedtissue-specifically, northern analysis was carried out. The HEIR-1 cDNAwas used first as a probe in order to investigate northern blotscontaining poly(A)+RNA from nine different adult human organs (FIG. 5A).Strong signals were obtained in RNA taken from lung, kidney and adrenalgland; this indicated that the gene in these tissues is transcribed inlarge amounts. In placenta, muscle, liver and pancreas the message waspresent only to a small degree. No transcript of the gene could bedetected in the brain. Two positive clones were obtained from a fetalbrain library. The sequence determined from one of the two clonesdiffered from the sequence shown in the sequence data insofar as thissequence contained an additional 100 nucleotides. Since these additionalnucleotides are located between two typical splice consensus sequences,the clone found would appear to be derived from RNA which has not beencompletely processed; however, one cannot rule out the possibility thatthis heterogeneity has a functional relevance. Thus, by definition, theHEIR-1 sequences according to the invention also include sequences whichdiffer from the ones shown in the sequence data by having additionalsequence sections which can be ascribed to alternative splicing.

The adrenal gland is the primary target organ for neuroblastoma tumours.More than 65% of tumours develop from cells of the medulla of theadrenal gland (Pochedly, 1976; Russell and Rubinstein, 1989). HEIR-1expression was investigated in medulla cells, once it had beenestablished that HEIR-1 is expressed strongly in the adrenal gland. Forthis purpose, the heir-1 probe was hybridised with northern blotscontaining poly(A)+RNA from cortex and medulla of the bovine adrenalgland. In preliminary tests zoo blot analysis had shown that HEIR-1 ishighly conserved in all mammalian species; bovine tissue proved assuitable as any other mammalian tissue. It was shown that HEIR-1expression in the adrenal gland is found predominantly in the medulla,whereas the cortex exhibits virtually no signal. This result was alsoconfirmed by positive signals from cultured chromaffin cells from themedulla of rat adrenal glands (PC12 cells).

In view of the high degree of homology between HEIR-1 and murine HLH 462and the easier availability of murine tissue compared with human tissue,the transcription pattern of the human probe was investigated in murinetissue in order to be able to develop a murine model for thetissue-specific function of the gene in humans. Investigation of aNorthern blot of poly(A)+RNAs from various murine tissues showed hightranscript concentrations in the lung, liver and kidney, whereas thetranscription in the adipose tissue, heart, spleen, brain and muscle wassignificantly less (FIG. 5B). No transcript could be detected in thetestes. The transcription patterns in the two different species werethus not entirely comparable; in particular, the different expression inthe liver showed that the two organisms did not have identicaldistribution of the gene product.

After it had been shown that the HEIR-1 gene is located in theneuroblastoma consensus deletion, transcription of HEIR-1 inneuroblastomas was investigated. Northern blots containing poly(A)+RNAfrom 12 different neuroblastoma cell lines were investigated with HEIR-1cDNA as probe. For this purpose the high expression of the gene in theadrenal glands was regarded as a normal control, as it is known thatmore than 65% of all neuroblastomas originate in this organ. As shown inFIG. 7, 10 of the tumour RNAs exhibited very weak HEIR-1 signals,indicating a specific reduction in the gene activity. Strong signalswere found only in the RNA of the tumour cell line SK-N-SH and asubclone thereof, SH-EP. In spite of the two different RNAconcentrations, all tumour RNAs showed bands of the same size as wereobserved in normal tissues. This indicates that the HEIR-1 mRNA shouldnot be affected by any tumour-specific structural rearrangement.

The amplification and overexpression of the oncogene N-myc ischaracteristic of numerous neuroblastomas, particulary at advancedstages (Zimmerman and Alt, 1990). In order to determine how the twodifferent levels of HEIR-1 expression are connected with the overexpression of N-myc, HEIR-1 was removed from the northern blots and theblots were again hybridised with the Nb-1 probe which is specific forthe human N-myc gene (Schwab et al., 1983). Of the 12 tumour cell lines,eight exhibited a very strong signal which indicated over expression ofN-myc (FIG. 7). When the HEIR-1 transcription was compared with the overexpression of N-myc in the relevant tumour cell lines, a clearcorrelation was found: those tumour cell lines which exhibited highconcentrations of N-myc had a very low level of HEIR-1 mRNA. In the twocell lines in which this correlation was not found, the low HEIR-1transcription possibly correlates inversely with an over expression ofc-myc, as the rehybridisation of the northern blot with a c-myc-specificprobe indicates (see FIG. 7); a clear correlation of HEIR-1 generallywith genes of the myc family still requires definitive confirmation.

Within the scope of the present invention it was further found that theexpression of HEIR-1 and N-myc are mutually exclusive in normaldevelopment: as for the proto-oncogene N-myc, it is known to beexpressed in normal embryonic development (Zimmerman et al., 1986).After it was shown by isolation of cDNA clones from the fetal brain thatHEIR-1 is expressed in embryos, investigations were carried out todiscover whether there might possibly be an inverse correlation betweenHEIR-1 and N-myc, not only tumour cells but in normal development aswell. For this purpose, both genes were analysed in their development byin situ hybridisation on murine tissue and compared.

The supposition that the HEIR-1 gene is involved in tumorgenesis wasthus confirmed by the investigations of HEIR-1 transcription in normaltissues and in neuroblastoma cells, carried out within the scope of thepresent invention, and by the observation that the HEIR-1 transcriptionlevels in human neuroblastomas and in normal tissues during developmentare inversely correlated with an over expression of N-myc.

According to a further aspect, the present invention relates to the useof HEIR-1 DNA sequences or parts thereof and antibodies against HEIR-1for the clinical diagnosis of pathological conditions which areassociated with an aberration in the neuroblastoma consensus deletionarea lp36.2-p36.12 or for diagnosing a predisposition to such diseases.By these diseases is meant primarily tumour diseases, particularlyneuroblastomas.

The fact that the HEIR-1 cDNA recognises an RFLP in Apa I-digestedgenomic DNA which makes it possible to identify chromosome 1 homologuesis the prerequisite for recognising allelic deletions which are specificto lp36.2-p36.12 and which occur, for example, in neuroblastomas.Compared with known RFLP probes (Fong et al., 1989; Weith et al., 1989)the information which is obtained with the DNA according to theinvention as probe is of great significance diagnosis of neuroblastoma,as an RFLP test based on the probe according to the invention willdetect a gene which is directly connected with the tumorigenesis. Withinthe scope of the tests carried out, a high degree of heterozygosity wasdetected in neuroblastoma patients; in view of this high degree ofheterozygosity a test based on the probe according to the invention ismore reliable than the known RFLP tests which operate with probes whichdetect a lower level of heterozygosity.

In the course of the RFLP tests carried out, it was found that the useof the complete coding region of the HEIR-1 cDNA gives reliable results.However, apart from the complete sequence, it is also possible to useparts thereof which recognise the RFLP. The suitability of cDNAfragments can be established by comparing any desired fragments, whichare preferably at least about 200 bp long for the purposes of obtaininga detectable signal, with the complete probe in terms of its ability torecognise the RFLP. Such fragments may, for example, be prepared by PCRamplification as a preliminary.

The use of the HEIR-1 cDNA probes according to the invention fordiagnosing lp36.2-p36.12 deletions in tumours is carried out accordingto standard methods: after the removal of a tumour, the DNA and inparallel thereto DNA from healthy tissue or peripheral blood from thepatient is prepared, cut with Apa I and fractionated by gelelectrophoresis. After Southern transfer onto membranes it is hybridisedwith the probe according to the invention (the probe need not correspondexactly to the cDNA sequence; it is sufficient if it corresponds to adegree which, under the particular conditions selected, makes itpossible to carry out hybridisation with the DNA, or possibly RNA whichis to be analysed). The probe may carry any desired labelling which canbe made visible and will not affect the hybridisation qualities of theprobe; examples of conventional labels are radioactivity as well asdigoxigenin or biotin in conjunction with fluorescence or alkalinephosphatase reaction.

The RFLP test for HEIR-1 deletion is conveniently carried out inparallel to the investigation into amplification of the N-myc gene.Alternatively to DNA, RNA from tissues which are to be investigated canbe analysed by means of the DNA according to the invention, e.g. bymeans of conventional northern blots.

Anti-HEIR-1 antibodies are used for primary diagnosis of pathologicalconditions associated with aberrations in the region lp36.2-p36.12 andin those cases where N-myc amplification and/or HEIR-1 deletion cannotbe detected or cannot be clearly shown. The assays using the antibodiesare generally conventional immunoassays in which the probe is intendedto form a binary or ternary complex between the HEIR-1 gene product orpossibly a fragment thereof with one or more antibodies, one of whichhas a detectable label. The use of an antibody with exclusivespecificity for HEIR-1, ie. an antibody which does not cross-react withHEIR-1 related proteins, makes it possible to determine HEIR-1specifically in tissues.

In the light of tests carried out recently (Bader et al., 1991) it isassumed that the chromosome 1p-Arm contains an element which has theability to revert the tumour phenotype into an untransformed phenotype.Since HEIR-1 maps into the neuroblastoma consensus deletion situated inthis region, it obviously satisfies one of the essential pre-requisitesfor a gene which is involved in the formation of neuroblastomas, on thebasis of its functional loss. A further indication that HEIR-1 isconnected both with differentiation and also with the tumorigenesis ofneuroblastomas arises from its expression pattern: in contrast to theadult brain, it is transcribed in the embryonic brain, which indicatesthat the gene is active in tissues which are engaged in development andtherefore contributes to processes of differentiation. Another essentialaspect of HEIR-1 genetic activity, on the basis of which it can beassumed that HEIR-1 acts as a tumour suppressor gene, is its strikingtranscription in the medulla of the adrenal gland, by contrast withminimal transcription in the majority of neuroblastomas. This fact isparticularly remarkable because the adrenal gland is the target tissueof most neuroblastomas. Since it is generally recognised that tumours atan advanced stage and their metastases originate predominantly frommedullary cells of the adrenal gland, it can be concluded from thepresent findings that the activity of HEIR-1 in tumours is specificallyreduced.

Six of the neuroblastoma cell lines investigated were examined in vivofor their ability to form tumours, 5 of them formed tumours inimmunodeficient mice. Comparison of the tumorigenicity with the HEIR-1expression shows a striking coincidence of normal genetic activity withthe inability to form tumours. From this it can be concluded that HEIR-1has an essential function in suppressing tumours in vivo.

The traces of transcripts detected which are found in the majority oftumour cell lines presumably result from the greatly reduced expressionof the alleles of the gene remaining the tumour cells. Earliercytogenetic and molecular investigations indicate that at least one copyof the region which includes HEIR-1 would appear to be retained in thetumour cell lines used (eg. GI-ME-N; Martinsson et al., 1989; N-16:Weith et al., 1989). It is not known at present whether regulation ofthe HEIR-1 transcription or post-transcriptional changes are responsiblefor the small amounts of HEIR-1 mRNA. To find out, the 5' controlregions of the gene are investigated.

Another indication of the involvement of HEIR-1 in neuroblastomatumorigenesis is provided by comparison of the HEIR-1 transcription withN-myc expression in neuroblastomas. It is known that N-myc isspecifically over-expressed in a high proportion of neuroblastomas at anadvanced stage and that it correlates positively with tumour progressionand metastases formation. Consequently, from the inverse correlationbetween HEIR-1 and N-myc activity, it can be deduced that HEIR-1 plays afunctional role in malignant transformation. The mechanism of thecorrelation has not yet been explained; however, in view of the presentresults, it would seem likeliest that the reduction in HEIR-1 expressionas a tumour-specific event precedes the N-myc over-expression and thisgene possibly negatively regulates the transcription of N-myc by an asyet unknown method. The structure of the gene, which is characteristicof a negatively regulated gene, would also be in keeping with such amechanism: without wishing to be tied down to this theory, thedimerisation of HEIR-1, occurring in healthy tissue, with a geneticproduct potentially acting as an oncogene could inhibit the latter. Theloss of the HEIR-1 gene could lead to constitive activation of theoncogene and hence initiate a tumour. Another indication of negativeregulation is provided by the mutual exclusion of the activity of thetwo genes in developing tissues, the results of the investigation of theembryonic fore-brain having been particularly suggestive: althoughHEIR-1 and N-myc are expressed at the same stage of development in thesame type of tissue, namely the neuroectoderm, they are active inseparate but adjoining areas. The mutual exclusion was particularlynoticeable because of sharp boundaries between the different expressingsections.

In order to confirm the function of HEIR-1 as a tumour suppressor gene,tests are carried out by means of which the influence of a normallyexpressed HEIR-1 gene (ie. corresponding, in terms of quantity andsequence, to the HEIR-1 expressed in healthy normal tissue) can beobserved in tumour cells. For expression analyses of this kind, tumourcell lines, eg. the neuroblastoma cell lines IMR-32, Vi856 and SK-N-SH,are transformed with vectors which are replicable in mammalian cells andcapable of being selected, containing the cDNA coding for HEIR-1 underthe control of a strong promoter, and investigated for their phenotype,particularly their malignancy. The loss of the malignant phenotypedefines the HEIR-1 gene as a tumour suppressor gene by means of whichpathological conditions which involve malfunction of the gene can betreated therapeutically. Such conditions include in particular thosetumour diseases which are diagnosed by means of the HEIR-1 probes. Theseare primarily neuroblastomas in which a direct correlation betweenHEIR-1 and tumorigenesis has been detected, as well as other tumourswhich have a corresponding abberation in chromosome lp36.2-p36.1 (thereare indications that other forms of cancer have significant chromosomalabberations in the same region of chromosome 1 as neuroblastoma, such ashepatoma, malignant melanoma, glioblastoma, Merkel's cell carcinoma andbreast cancer). Confirmation of the fact that HEIR-1 is involved in theformation of these tumours can be obtained by carrying out theinvestigations which were performed for neuroblastoma using HEIR-1,including the functional expression analyses, in cells originated fromthe tumours in question.

The principle of therapeutic treatment consists in administering atherapeutically effective quantity of HEIR-1 to the body or causing itto be expressed in the body, i.e. supplying the body with the geneticproduct or, in the course of gene therapy, the gene, e.g. within thescope of whole body treatment. The gene as part of a vector under thecontrol of a tissue-specific promoter is introduced into the body usinggene transfer procedures, e.g. using ligands which bind totissue-specific receptors (a method of this kind which uses transferrinconjugates with DNA for receptor-mediated endocytosis is described in EP388 758) and is expressed in the target tissue. In the treatment ofneuroblastomas, suitable gene transfer agents are ligands for receptorswhich are expressed by cells originating from the neural strip, e.g.transferrin (experiments have shown that DNA associated with transferrinis efficiently taken up into such cells). Since the transcriptionpatterns obtained in the course of the present invention have showntissue specificity in HEIR-1 transcription, basically tissue specificityis sought within the scope of gene therapy. This is also true when usingretroviral vectors in which the tissue specificity of the gene constructused is of major importance, particularly as a result of possibledominance of the retroviral promoter. Conveniently, the HEIR-1 promoteris used for gene constructs for use in gene therapy, in order toachieve, as far as possible, tissue specificity and the amount of HEIR-1expression corresponding to expression in normal tissue. To achieve moreintense expression, multiple copies of promoter sub-fragments maypossibly be contained in the construct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. FIG. 1A shows the pulse field gel hybridization ofmicroclone p1-112B. FIG. 1B is a schematic representation of the genomicregion characterized by p1-112B. The probe is illustrated by a shadedhorizontal bar.

FIG. 2: Restriction map of cosmid C1-112B.

FIGS. 3A and 3B. FIG. 3A shows the nucleotide sequence of the cDNA cloneknown as HEIR-1 (SEQ ID NO: 1). FIG. 3B shows a comparison of thederived amino acid sequences of HEIR-1 (SEQ ID NO: 2) with murine HLH462(SEQ ID NO: 4) protein.

FIG. 4: Amino acid sequence comparison of the HLH motif between HEIR-1(SEQ ID NO:7), murine HLH562 (SEQ ID NO:7), murine Id (SEQ ID NO:8) anddrosophila emc (SEQ ID NO:9). The amino acids conserved in all 4proteins are shown in boxes.

FIGS. 5A, 5B, and 5C. FIG. 5A shows northern blots of poly(A)+RNA from 9different adult tissues hybridized to a HEIR-1 cDNA probe. FIG. 5B showsnorthern blots of poly(A)+RNA from adult mouse tissue, hybridized with ahuman HEIR-1 probe. FIG. 5C shows a northern hybridization of the HEIR-1cDNA probe with poly(A)+RNAs of cortex of bovine adrenal gland, medullaof bovine adrenal gland and rat PC12 cells.

FIGS. 6A, 6B and 6C. FIG. 6A shows a Southern blot of EcoRI-digested DNAfrom different (MouseXhuman) microcell hybrids hybridized with a ³²P-labeled HEIR-1 cDNA. FIG. 6B shows an analysis of the loss ofheterozygosity (LOH) at the HEIR-1 locus of the neuroblastoma patientN-29. FIG. 6C shows a diagram demonstrating the localization of theHEIR-1 gene in the neuroblastoma consensus deletion.

FIG. 7: Transcription of HEIR-1, N-myc and c-myc in neuroblastoma celllines.

FIG. 8: LOH-analysis (Loss of Heterozygosity) of two neuroblastomatumours (N-15, N-29) with the probe HEIR-1.

FIG. 9: Western-Blot Analysis. Detection of recombinant HEIR-1 by meansof polyclonal antibodies.

The invention is illustrated by means of the Examples which follow:

Materials and Methods

Cell lines and materials obtained from neuroblastoma patients.

The following neuroblastoma cell lines were used: Vi856; N15, N16 (Weithet al., 1989), SK-N-SH, SH-EP (Biedler et al., 1973), GI-ME-N (Donti etal., 1988), GI-LI-N, GI-CA-N (Longo et al., 1988), LS (Rudolph et al.,1991), Kelly (Schwab et al., 1983), IMR-32 (Tumilowicz et al., 1970),NMB (Balaban-Malenbaum and Gilbert, 1977), LAN-5 (eg. Minth and Dixon,1990). All the cell lines were kept in RPMI with 10% FCS, 1%penicillin/streptomycin and 4 mM L-glutamine. The hybrid (MouseXHuman)cell lines 8:4-BE1, 20-EA3, 20-DH8 and 20-AE2 have been described byMartinsson et al., 1989. Rat PC 12 cells (Greene and Tischler, 1976)were grown in DMEM containing 15% equine serum, 1%penicillin/streptomycin and 4 mM L-glutamine. Tumour tissue andperipheral blood taken from patients designated N-15 and N-29 have beendescribed by Weith et al. (1989).

DNA Probes

p1-112B is a 2.5 kbp long genomic DNA clone from a 1pter-p35-specificmicroclone bank (Martinsson et al., 1989). The original microclone wasprepared by cloning Eco RI fragments into the lambda vector NM1149. Forfurther use the fragment was recloned into the plasmid vector BluescriptKS+ (Stratagene). p1-112B was mapped by Southern hybridisation on aseries of (MouseXHuman) hybrid cell lines in the region 1pter-p36.12.C1-112B represents a cosmid clone isolated from a genomic cosmid library(see below) by means of the probe p1-112B. The insert of C1-112B isabout 37 kbp long (FIG. 2). C1-112B/9.ORI is a 9.0 kbp Eco RIsub-fragment from the cosmid C1-112B (FIG. 2). In order to prepare thisclone, 8 μg of C1-112B DNA was digested with the restriction enzyme EcoRI (Boehringer Mannheim) in accordance with the manufacturersinstructions, fractionated by electrophoresis in a 0.6% agarose-gel andthe DNA was revealed by ethidium bromide staining. An agarose fragmentwhich contained the 9.0 kbp sub-fragment of the cosmid was excised andthe DNA contained was isolated from the agarose by phenol extraction. Nomeasurement of the concentration of the DNA obtained was carried out.The DNA was then ligated with 50 ng of Eco RI digested de-phosphorylatedplasmid DNA (Bluescript KS+) in 10 μl volume in the presence of 1 unitof T4-DNA ligase (Boehringer Mannheim) and ligation buffer in accordancewith the manufacturer's instructions. Ligation was carried out for 14hours at 12° C. Then the entire ligation mixture was used to transformXL-1blue host bacteria (Stratagene). Transformation was carried outusing the standard method (Sambrook et al., 1989). After the cells hadbeen plated out onto LB-amp (LB-medium+ampicillin [50 μg/ml]) plates andincubated overnight, recombinant colonies could be isolated. Nb-1 is anNy-myc specific genomic 1.0 kbp EcoRI/BamHI fragment, cloned in pBR322(Schwab et al., 1983). The c-myc specific clone pMc-myc 54 is a 1.3 kbpcDNA fragment cloned in PSP64 (Darveau et al., 1985).

Radioactive labelling of DNA and cDNA probes

Insert fragments of the recombinant clones were used as probes forhybridisation. 5 to 8 μg of a cloned DNA were digested with restrictionenzymes to separate the insert from the vector DNA, fractionated byagarose gel electrophoresis and the insert fragment in an agarosefragment was separated out. Then the DNA was separated from the agaroseby phenolisation. 20-50 ng of this insert DNA were labelled with32p-dCTP by "random priming" using the standard method (Sambrook et al.,1989) and used as hybridisation probes.

Analysis of genomic DNA

Genomic DNA from whole blood and various organ and tissue samples wasisolated using standard methods (Sambrook et al., 1989), digested withrestriction endonucleases in accordance with the manufacturer'sinstructions (Boehringer Mannheim, New England Biolabs, Promega) andfractionated by gel electrophoresis in 0.8% agarose gels. This wasfollowed by partial depurination, denaturation and capillary transferonto nylon membranes (GeneScreen plus, NEN) under denaturing conditionsin accordance with established methods (Sambrook et al., 1989). Themembranes were air-dryed after the transfer of the DNA, incubated at 80°C. for 30 minutes and subjected to UV irradiation. For hybridisation,membranes were first pre-hybridised in 500 mM Na-phosphate buffer, pH7.2, 7% SDS and 1 mM EDTA (68° C., 1-2 hours) and then incubated at 68°C. overnight in the same buffer with the probe, with constant agitation.The membranes were generally scrupulously washed (40 mM Na-phosphatebuffer pH 7.2, 1% SDS, 68° C.), sealed in PE film whilst still damp andexposed on X-ray film (Kodak XAR-5) with intensifier film.

RNA Analyses

Total RNA from tissues and from cell culture material was isolated usingthe guanidium-thiocyanate method described by Chomczynski and Sacchi(1987). Poly(A)+-RNA was isolated using the conventional method(Sambrook et al., 1989) by affinity chromatography using oligo-d(T)columns. Poly(A)+RNA was isolated from about 2-5 mg of total RNA in eachcase. The yield was about 2-3% of the total RNA. For northern analysis,from each tissue, 2 μg (normal human tissue) or 3 μg (mouse tissue andneuroblastoma culture cells) were fractionated in formaldehyde-agarosegels using the appropriate methods (Sambrook et al., 1989) andtransferred onto membranes (GeneScreen, NEN). The northern blots werehybridised using the same method as for Southern blots.

DNA Sequence Analysis

The dideoxy sequencing kit obtainable from Applied Biosystems was used.

EXAMPLE 1

Pulsed Field Gel Electrophoresis

High molecular weight genomic DNA was isolated from human sperm orlymphocytes which had been enclosed in agarose blocks, using publishedmethods (Herrmann et al., 1987). The DNA in the agarose blocks (about 10μg/block) was digested with methylation-sensitive, rarely cuttingrestriction endonucleases (2-30 units per block) in accordance with therecommendations of the manufacturer (New England Biolabs, BoehringerMannheim) by either single or double digestion. Then the agarose blockswith the digested DNA were separated in 1% agarose gels by CHEFelectrophoresis in a pulsating electrical field. A Pulsaphor system(LKB) was used for this. DNA separation was carried out in 3-phase runs:Phase 1: 40 s (8 h), Phase 2: 15 s (8 h), Phase 3: 3 s (8 h).Electrophoresis was carried out in 0.5×Tris-borate-EDTA (TBE, seeSambrook et al., 1989) at 200 V and 10° C. Subsequent Southern transferand hybridisation of membranes were carried out as described above.

The results of the PFGE are shown in FIG. 1: A: 32p-labelled p1-112B washybridised with a PFG blot which contained about 10 μg of normal humansperm DNA in each trace, digested with the enzymes mentioned. Thefragment which represents the spacing between the two CpG Islands ismarked by an arrow. The sizes of the lambda-DNA concatamers are shown inkbp as markers. The autoradiography exposure took 3 days. B: Schematicrepresentation of the genomic region characterised by p1-112B. The probeis illustrated by a shaded horizontal bar. Two CpG Islands are indicatedby a cluster of vertical bars. Additional bands detected with the probe(eg. traces NarI and SmaI in A, shown as a vertical bar in B) originatefrom recognition sites within the probe and represent fragments outsidethe section between the CpG Islands.

EXAMPLE 2

a) Preparing a cosmid library

High molecular weight genomic DNA was isolated from human lymphocytes:the erythrocytes were lysed from 40 ml of peripheral blood and the DNAwas extracted from the lymphocytes in 20 mM tris-HCl, pH 7.6, 20 mMEDTA, 1% sarkosyl and 200 μg/ml proteinase K (Boehringer Mannheim). TheDNA obtained was partially digested with the restriction enzyme Mbo I,without previous phenolisation, so that on average 35-45 kbp longfragments were obtained. A concentration of fragments of this length wasachieved by gel fractionation in 0.25% agarose gels.

Then the genomic DNA fragments and Bam H1-cut, dephosphorylatedvector-DNA (cosmid vector pWE15, Stratagene) were ligated together in aweight ratio of 1:5. In order to do this, 5 μg of genomic fragments and25 μg of vector DNA in a total 10 μl volume were incubated for 16 hoursat 12° C. in the presence of T4-DNA ligase and ligation buffer(Boehringer Mannheim). The ligated material was packaged with anin-vitro packaging mix in infectious phage particles and used totransform E. coli NM554 cells. A yield of 2×10⁷ recombinants per μg ofgenomic DNA was achieved.

2.5-2.8×10⁷ recombinant bacteria from the cosmid library were plated outon LB^(amp) plates measuring 22×22 cm directly on membranes (GeneScreenPlus, NEN) and incubated for 13 hours. Two copies (replica filters) wereprepared from the primary colony membranes (master filters) andincubated for a further 8 hours for colony growth. The master filterswere then impregnated with freezing medium (LB+20% glycerol), placed onPlexiglass® plates and stored at -80° C. Replica filters wereimpregnated for 5 minutes with 0.2M NaOH, 1% SDS, 1.5M NaCl, thenneutralised in 50 mM Na-phosphate buffer, pH 6.5, and air-dried.

b) Screening of the cosmid library using the probe p1-112B

Insert DNA from p1-112B was labelled by random priming (see above) andhybridised as a probe on the replica filters of the cosmid library understandard conditions (Sambrook et al., 1989). Areas with positivecolonies were excised accordingly from the deep-frozen master filters,resuspended in LB medium and plated out on filters at low density(approximately 600-1000 colonies per 85 mm of filter), incubated, andhybridised again with labelled p1-112B probe. After this step, positivesignals could be attributed to individual colonies. The individualcolonies were isolated and amplified in 50 ml cultures for the isolationof cosmid DNA. Cosmid DNA was prepared using standard methods (Sambrooket al., 1989).

EXAMPLE 3

Isolation of a C1-112B/9.ORI positive cDNA from a HeLa cDNA library

140000 plaques from a HeLa cDNA library (Stratagene) were searched forpositive cDNA plaques using the genomic sub-fragment C1-112B/9.ORI. Theprinciple working steps for isolating cDNA clones from a cDNA libraryare shown as follows.

A. Primary Screening:

1. Plating out the phages.

The phages were diluted with TM in accordance with the initial titre(1.5×10¹⁰ pfu/ml) so as to obtain roughly 70000 plaques per 22×22 cm²plate. The corresponding quantity of phages was then incubated with 4 mlof an overnight culture of XL-1 blue bacteria for 20 minutes at 37° C.Then 40 ml of top agar (preheated to 55° C.) was added to this mixtureand it was plated out on an LB plate measuring 22×22 cm². It was thenincubated overnight at 37° C.

2. Plaque transfer onto GeneScreen plus membranes. After overnightincubation has occurred followed by storage at 4° C. for 2 hours, theplate was covered for 2 minutes with a 21×21 cm² GeneScreen plusmembrane. Then the membrane was placed for 3 minutes on a filter paperwhich had been impregnated with denaturing solution (1.5M NaCl, 0.5MNaOH). It was then incubated for 10 minutes on a filter paperimpregnated with neutralising solution. After drying in air for 1 hourand at 80° C. for 30 minutes, the DNA was fixed on the membrane by UVirradiation.

3. Hybridisation of the membranes with C1-112B/9.ORI. This step wascarried out using conventional methods (Sambrook et al., 1989).

B. Secondary Screening:

Since individual plaques could not be achieved because of the highdensity (70000 plaques per plate), secondary screening had to be carriedout (with the purpose of isolating individual plaques).

1. Isolation of positive plaques.

The plaques which had produced positive signals in the primary screeningwere isolated from the large plate. In accordance with the methoddescribed in A, secondary screening was carried out. The onlydifferences in this screening related to the size of the plates (8 cmPetri dishes) and the density of plaques per dish (150 plaques per dish;different dilutions of the positive plaque were plated out).

2. Analysis of positive individual plaques.

This was carried out using the methods currently used in laboratories,eg. in vivo excision, carried out in accordance with the instructions ofStratagene, PCR analysis, (Sambrook et al. 1989).

The cDNA clone known as HEIR-1 was obtained, which has the sequenceshown in SEQ ID NO: 1. The cDNA sequence obtained is also shown in FIG.3A; the two start codons, a polyadenylation signal and the transcriptiontermination codon are underlined and an ATTTA motif is marked by adashed line. FIG. 3B shows a comparison of the derived amino acidsequence of HEIR-1 with the murine HLH462 protein.

C. Direct sequencing of the 5' regulatory region: The sequence of thepromoter was obtained by sequencing the clone C1-112B/9ORI. Theoligonucleotide TGG GGA GTG AGT CCA GAG, shown in SEQ ID NO: 6, which iscomplementary to the HEIR-1 cDNA, was used as sequencing primer.

EXAMPLE 4

Transcription of the HEIR-1 gene in various human tissues

The results of the northern hybridisations, carried out as describedunder "Materials and Methods", are shown in FIG. 5.

FIG. 5A: Northern blots of poly(A)+RNA (2 μg) from 9 different adulttissues. The 8 traces on the left represent a so-called MTN (multipletissue northern), obtainable from Clontech (Palo Alto). For isolatingadrenal-RNA, tissue was used which had been taken from a patient in thecourse of a kidney and adrenal operation. The size of the positive bandis given in kb. The calibration of the quantity of RNA applied to eachtrace was carried out by hybridising aglyceraldehyde-phosphate-dehydrogenase probe (GAPDH) with the blot afterremoving the HEIR-1 probe (shown at the bottom of the Fig.). Theautoradiography exposure took 1 day (HEIR-1) or 4 hours (calibration).

FIG. 5B: Northern blots of poly(A)+RNA (3 μg) from adult mouse tissue,hybridised with human HEIR-1, as described under A.

FIG. 5C: Northern hybridisation of the HEIR-1 cDNA probe withpoly(A)+RNAs (for 3 μg) of cortex of bovine adrenal gland, medulla ofbovine adrenal gland and rat PC12 cells. The quantities of RNA werestandardised by methylene blue staining of RNA on the filter and with arat GAPDH probe. The size of the positive band is given in kb. Theautoradiograph was exposed for 21/2 days (HEIR-1 on bovine RNA) and 1day (HEIR-1 on PCT12 cells).

EXAMPLE 5

Transcription of HEIR-1 and c-myc in neuroblastoma cell lines

Northern blots containing poly(A)+RNA (3 μg/trace) from 12 differentneuroblastoma cell lines were hybridised one after the other with the 3cDNA probes specified. Calibration was carried out using the GAPDH probe(bottom trace). The autoradiography exposure took 5 to 18 hours (FIG.7).

EXAMPLE 6

Inverse correlation between the expression of HEIR-1 and N-myc inembryonic mouse tissue

The preparation of embryonic tissue sections and in situ hybridisationswere carried out as described by Aguzzi et al., 1990. Sense andanti-sense cRNA probes were prepared by in vitro transcription of theHEIR-1 cDNA or of a mouse sub-clone corresponding to the third exon ofN-myc (DePinho et al., 1986), in the presence of 35S-labelled rUTP.Sense-transcribed probes were used as control.

In situ hybridisation of sections of mouse tissue embedded in paraffin,from various embryonic stages, which had been hybridised either withHEIR-1 or N-myc anti-sense probes, exhibited clearly tissue-specificsignals for both. In particular, the expression of each of the genes inthe developing brain was limited to various sections of theneuroectoderm. In the layer of forebrain neuroectodermal cells, N-mycwas expressed predominantly in the regions of the cortex andhypothalamus. However, a sharply defined ventral part of theneuroectoderm, which comprises the cordal telencephalon and itsconnections to the developing diencephalon, was completely free fromN-myc message. By contrast, this was the only part of the forebrainneuroectoderm which showed HEIR-1 expression. The regions which showedeither HEIR-1 or N-myc expression were separated by strikingly sharp andessentially coinciding boundaries. At the boundary between theneuroectoderm and tissues of mesodermal origin there was alsocomplementary expression of the two genes: HEIR-1 was strongly expressedin the developing skull structures, whereas N-myc in this tissueexhibited only non-specific background values.

EXAMPLE 7

Identification of a restriction fragment length polymorphism (RFLP) forthe probe HEIR-1

Genomic DNA was isolated from peripheral blood from 7 differentindividuals using standard methods (Sambrook et al., 1989). Thecollection of DNAs was digested with 40 different restrictionendonucleases (Boehringer Mannheim, New England Biolabs, Promega) inaccordance with the manufacturer's instructions and fractionated by gelelectrophoresis in 0.8% agarose gels. Southern transfer of thefractionated DNA onto GeneScreen membranes (NEN) was carried out usingthe standard methods described above. The membranes were hybridised withthe ³² P-dCTP-labelled probe heir-1 in accordance with the standardmethods mentioned, washed under conditions of high stringency (40 mMNa-phosphate buffer, pH 7.2, 1% SDS, 68° C.) and exposed for 24 hours onKodak X-AR5 film. An RFLP was recognised by comparison of thehybridisation patterns on DNAs which had been digested with the sameenzyme. The band pattern on Apa I-digested DNAs showed the occurrence oftwo alleles in different individuals. The size of the alleles, constantbands and allele frequency are shown in Table 1:

                  TABLE 1                                                         ______________________________________                                        RFLP for the locus HEIR-1:                                                    ______________________________________                                        Fragments (kbp)                                                                         1.0    0.6    12.0  6.5  3.2  Frequency*                            Allele A1 +      -      +     +    +    57%                                   Allele A2 -      +      +     +    +    43%                                   ______________________________________                                         *Total frequency determined after examining a total of 56 chromosomes    

EXAMPLE 8

Localisation of HEIR-1 in the neuroblastoma consensus deletion

A Southern blot of EcoRI-digested DNA from different (MouseXhuman)microcell hybrids and the relevant human and mouse controls washybridised with ³² P-labelled HEIR-1-cDNA. The Southern blot is shown inFIG. 6.

A: Human (10 μg): Lymphocyte DNA; Mouse (20 μg): genomic Balb/c-DNA.Hybrids (20 μg each): 8:4-BE1, total human chromosome 1; 20-EA3,del(1)(pter-p36.12); 20-DH8, del(1)(pter-p31); 20-AE2, del(1)(pter-p11).Solid vertical bars in the bottom figure show the chromosome 1 materialwhich is contained in the hybrids, whereas shaded regions show thedeletions. The probe showed a human (H) and a murine (M) band in therelevant traces.

B: Analysis of the loss of heterozygosity (LOH) at the HEIR-1 locus ofthe neuroblastoma patient N-29. A Southern blot containing ApaI-digestednormal DNA from peripheral blood (N) and tumour (T) DNA was hybridisedwith the HEIR-1 probe. Normal DNA shows two bands at 1 kbp and 0.6 kbpwhich correspond to the two HEIR-1 alleles. The tumour DNA shows theloss of the upper allele (arrow). The consensus deletion (cons.del.) isshown as the region between the proximal break-off point of the deletionin 20-EA3 and the distal break-off point of the allelic N-29 deletion.The autoradiography exposure took 16 hours (A) and 4 days (B).

EXAMPLE 9

Loss of heterozygosity analysis in neuroblastoma tumours

10 μg of genomic DNA from tumour tissue and corresponding normal tissue(peripheral blood) from neuroblastoma patients, prepared as described inExample 1, was cut with ApaI in accordance with the manufacturer'sinstructions (Boehringer Mannheim), fractionated in 0.8% agarose gels,transferred to nylon membranes and hybridised with the ³² P-labelledHEIR-1 probe. Then the filters were scrupulously washed as described inExample 1 and exposed on X-ray film. The RFLP is illustrated in FIG. 8and shows the allele pattern described in Example 7 for tworepresentative neuroblastoma patients. The residual hybridisation whichis visible in the tumour DNA of N-15 results from the fact that thestarting material used to isolate the tumour DNA contained traces ofnormal tissue.

EXAMPLE 10

Expression of recombinant HEIR-1 in E. coli

The E. coli expression plasmid pET-2a described by Studier et al., 1990was modified by replacing the short Ndel-BamHI fragment by anoligonucleotide coding 6 histidine amino acids (Adams et al., 1992). Inthe 3' position relative to this oligonucleotide an HEIR-1 coding PCRfragment corresponding to the sequence shown in SEQ ID NO: 3 was cloned,taking into account the reading frame. Consequently, this bacterialexpression vector (pETH-2a HEIR-1) contains sequences which code for a(His) 6x-HEIR-1 fusion protein. After transformation of the E. colistrain BL21 (DE3) the protein was expressed in E. coli by induction withIPTG in accordance with the standard procedure (Sambrook et al.) andafter bacterial lysis by means of nickel complex affinity chromatography(Hochuli et al., 1988) it was isolated from the bacterial extract.

EXAMPLE 11

Preparation of polyclonal antibodies against HEIR-1

In order to obtain antibodies a rabbit was immunised with roughly 300 μgof HEIR-1 protein (emulsified with complete Freund's adjuvant). After 2and 5 weeks, the rabbit was immunised with approximately 100 μg HEIR-1protein (emulsified with incomplete Freund's adjuvant) to increase theantibody concentration (booster). The serum isolated from the immunisedrabbit was tested by Western blot analysis (Sambrook et al.) as shown inFIG. 9. Detection of the protein-bound antibody on the Western blot wascarried out using ECL analysis (Enhanced Chemiluminescence, Amersham).In FIG. 9, the traces of the blot designated 1 and 2 represent the totalbacterial protein before (1) and after (2) induction with IPTG. Trace 3contains purified recombinant HEIR-1 fusion protein.

Bibliography

Adams, B., et al., 1992, Genes & Dev. 6, 1589-1607.

Aguzzi, A., Wagner, E. F., Williams, L. R. and Courtneidge, S. A., 1990,The New Biologist 2, 533-543.

Bader, S. A., Fasching, C., Brodeur, G. M. and Standbridge, E. J., 1991,Cell Growth and Diff. 2, 245-255.

Balaban-Malenbaum, G. and Gilbert, F., 1977, Science 198, 739-741.

Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. and Weintraub,H., 1990, Cell 61, 49-59.

Bernard, O., Cory, S., Gerondakis, S., Webb, E. and Adams, J. M., 1983,Embo J. 2, 2375-2383.

Biedler, J. L., Helson, L. and Spengler, B. A., 1973, Cancer Res. 33,2643-2652.

Bird, A. P., 1986, Nature 321, 209-213.

Brawerman, G., 1989, Cell 57, 9-10.

Chomczynsky, P. and Sacchi, N., 1987, Anal. Biochem. 162, 156-159.

Christy, B. A., Sanders, L. K., Lau, L. F., Copeland, N. G., Jenkins, N.A. and Nathans, D., 1991, Proc.Nat.Acad.Sci. USA 88, 1815-1819.

Darveau, A., Pelletier J. and Sonnenberg, N., 1985, Proc.Nat.Acad.Sci.USA 82, 2315-2319.

Davis, R. L., Weintraub, H. und Lassar, A. B., 1987, Cell 51, 987-1000.

DePinho, R. A., Legouy, E., Feldman, L. B., Kohl, N. E., Yancopoulos, G.D. and Alt, F. W., 1986, Proc.Natl.Acad.Sci. USA 83, 1827-1831.

Donti, E., Longo, L., Tononi, G. P., Verdona, G., Melodia, A., Lanino,E. and Coraglia-Ferraris, P., 1988, Cancer Genet. Cytogenet. 30,225-231.

Ellis, H. M., Spann, D. R. and Posakony, J. W., 1990, Cell 61, 27-38.

Fong, C. T., Dracopoli, N. C., White, P. S., Merril, P. T., Griffith, R.C., Housman, D. E. and Brodeur, G. M., 1989, Proc.Natl.Acad.Sci. USA 86,3753-3757.

Friend, S. H., Bernards, R. Rogelij, S., Weinberg, R. A. Papaport, J.M., Albert, D. M. and Dryja, T. P., 1986, Nature 323, 643-646.

Garell, J. and Modolell, J., 1990, Cell 61, 39-48.

Ghysen, A. and Dambly-Chaudiere, C., 1989, Trends in Genetics 5,251-255.

Greene, L. A. and Tischler, A. S., 1976, Proc.Natl.Acad.Sci. USA 73,2424-2428.

Harlow, E. and Lane, D., 1988, Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory.

Herrmann, B. G., Barlow, D. P. and Lehrach, H., 1987, Cell 48, 813-825.

Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. and Stuber, D., 1988,Bio/Technology 6, 1321-1325.

Jones, N., 1990, Cell 61, 39-48.

Kohler, G. and Milstein, C., 1975, Nature 265, 495-497.

Longo, L., Christiansen, H., Christiansen, N. M., Cornaglia-Ferraris, P.and Lampert, F., 1988, J. Cancer Res. Clin. Oncol. 114, 636-640.

Luscher, B. and Eisenmann, R. N., 1990, Genes Dev. 4, 2025-2035.

Martinsson, T., Weith, A., Cziepluch, C. and Schwab, M., 1989, GenesChrom. Canc. 1, 67-78.

Minth, C. D. and Dixon, J. E., 1990, J. Biol. Chem. 265, 12933-12939.

Murre, C., Schonleber McCraw, P. and Baltimore, D., 1989a, Cell 56,777-783.

Murre, C., Schonleber McCraw, P., Vaessin, H., Caudy, M., Lan, L. Y,Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, A.B., Weintraub, H. and Baltimore, D., 1989b, Cell 58, 537-5443.

Pochedly, C., 1976, In Pochedly, C. (ed.) Neuroblastoma, Edward ArnoldLtd., London, pp. 1-34.

Rudolph, G., Schilbach-Stuckle, K., Handgretinger, R., Kaiser, P. andHameister, H., 1991, Hum. Genet. 86, 562-566.

Russell, D. S. and Rubinstein, L. J., 1989, Pathology of Tumors of theNervous System, 5th edition, Edward Arnold Publ., London, Melbourne,Auckland.

Sambrook, J., Fritsch, E. F. and Maniatis, T., 1989, Cold Spring HarborLaboratory Press (2.Auflage).

Schwab, M., Alitalo, K., Klempnauer, K. H., Varmus, H. E., Bishop, J.M., Gilbert, F., Brodeur, G., Goldstein, M. and Trent, J., 1983, Nature305, 245-248.

Studier, W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W., 1990,Methods Enzymol. 185, 60-89.

Tumilowicz, J. J., Nichols, W. W., Cholon, J. J. and Greene, A. E.,1970, Cancer Res. 30, 2110-2118.

Weinberg, R. A., 1991, Science 254, 1138-1146.

Weith, A., Martinsson, T., Cziepluch, C., Bruederlein, S., Amler, L. C.,Berthold, F. and Schwab, M., 1989, Genes, Chrom. Canc. 1, 159-166.

Zimmerman, K. A., Yancopoulos, G. D., Collum, R. G., Smith, R. K., Kohl,N. E., Denis, K. A., Nau, M. N., Witte, O. N., Toran-Allerand, D., Gee,C. E., Minna, J. D. and Alt, F. W., 1986, Nature 319, 780-783.

Zimmerman, K. and Alt, F. W., 1990, Critical Reviews in Oncogenesis 2,75-95.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 982 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGACAATTTTCAGCAGGAAGAAGTAGAAAGGATAAAATGGATCCTGCACCACGG54                      MetAspProAlaProArg                                                            15                                                                            GAACCTCACAGCACCTCACTTCTTTTGGTTTTCTTTCTCTTTGGGGCA102                           GluProHisSerThrSerLeuLeuLeuValPhePheLeuPheGlyAla                              101520                                                                        CCTCTGGACTCACTCCCCAGCATGAAGGCGCTGAGCCCGGTGCGCGGC150                           ProLeuAspSerLeuProSerMetLysAlaLeuSerProValArgGly                              253035                                                                        TGCTACGAGGCGGTGTGCTGCCTGTCGGAACGCAGTCTGGCCATCGCC198                           CysTyrGluAlaValCysCysLeuSerGluArgSerLeuAlaIleAla                              404550                                                                        CGGGGCCGAGGGAAGGGCCCGGCAGCTGAGGAGCCGCTGAGCTTGCTG246                           ArgGlyArgGlyLysGlyProAlaAlaGluGluProLeuSerLeuLeu                              55606570                                                                      GACGACATGAACCACTGCTACTCCCGCCTGCGGGAACTGGTACCCGGA294                           AspAspMetAsnHisCysTyrSerArgLeuArgGluLeuValProGly                              758085                                                                        GTCCCGAGAGGCACTCAGCTTAGCCAGGTGGAAATCCTACAGCGCGTC342                           ValProArgGlyThrGlnLeuSerGlnValGluIleLeuGlnArgVal                              9095100                                                                       ATCGACTACATTCTCGACCTGCAGGTAGTCCTGGCCGAGCCAGCCCCT390                           IleAspTyrIleLeuAspLeuGlnValValLeuAlaGluProAlaPro                              105110115                                                                     GGACCCCCTGATGGCCCCCACCTTCCCATCCAGACAGCCGAGCTCGCT438                           GlyProProAspGlyProHisLeuProIleGlnThrAlaGluLeuAla                              120125130                                                                     CCGGAACTTGTCATCTCCAACGACAAAAGGAGCTTTTGCCACT481                                ProGluLeuValIleSerAsnAspLysArgSerPheCysHis                                    135140145                                                                     GACTCGGCCGTGTCCTGACACCTCCAGAACGCAGGTGCTGGCGCCCGTTCTGCCTGGGAC541               CCCGGGAACCTCTCCTGCCGGAAGCCGGACGGCAGGGATGGGCCCCAACTTCGCCCTGCC601               CACTTGACTTCACCAAATCCCTTCCTGGAGACTGAACCTGGTGCTCAGGAGCGAAGGACT661               GTGAACTTGTGGCCTGAAGAGCCAGAGCTAGCTCTGGCCACCAGCTGGGCGACGTCACCC721               TGCTCCCACCCCACCCCAAGTTCTAAGGTCTTTTCAGAGCGTGGAGGTGTGGAAGGAGTG781               GCTGCTCTCCAAACTATGCCAAGGCGGCGGCAGAGCTGGTCTTCTGGTCTCCTTGGAGAA841               AGGTTCTGTTGCCCTGATTTATGAACTCTATAATAGAGTATATAGGTTTTGTACCTTTTT901               TACAGGAAGGTGACTTTCTGTAACAATGCGATGTATATTAAACTTTTTATAAAAGTTAAC961               ATTTTGCATAATAAACGATTT982                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 148 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAspProAlaProArgGluProHisSerThrSerLeuLeuLeuVal                              151015                                                                        PhePheLeuPheGlyAlaProLeuAspSerLeuProSerMetLysAla                              202530                                                                        LeuSerProValArgGlyCysTyrGluAlaValCysCysLeuSerGlu                              354045                                                                        ArgSerLeuAlaIleAlaArgGlyArgGlyLysGlyProAlaAlaGlu                              505560                                                                        GluProLeuSerLeuLeuAspAspMetAsnHisCysTyrSerArgLeu                              65707580                                                                      ArgGluLeuValProGlyValProArgGlyThrGlnLeuSerGlnVal                              859095                                                                        GluIleLeuGlnArgValIleAspTyrIleLeuAspLeuGlnValVal                              100105110                                                                     LeuAlaGluProAlaProGlyProProAspGlyProHisLeuProIle                              115120125                                                                     GlnThrAlaGluLeuAlaProGluLeuValIleSerAsnAspLysArg                              130135140                                                                     SerPheCysHis                                                                  145                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGAAGGCGCTGAGCCCGGTGCGCGGCTGCTACGAGGCGGTGTGCTGC48                            MetLysAlaLeuSerProValArgGlyCysTyrGluAlaValCysCys                              151015                                                                        CTGTCGGAACGCAGTCTGGCCATCGCCCGGGGCCGAGGGAAGGGCCCG96                            LeuSerGluArgSerLeuAlaIleAlaArgGlyArgGlyLysGlyPro                              202530                                                                        GCAGCTGAGGAGCCGCTGAGCTTGCTGGACGACATGAACCACTGCTAC144                           AlaAlaGluGluProLeuSerLeuLeuAspAspMetAsnHisCysTyr                              354045                                                                        TCCCGCCTGCGGGAACTGGTACCCGGAGTCCCGAGAGGCACTCAGCTT192                           SerArgLeuArgGluLeuValProGlyValProArgGlyThrGlnLeu                              505560                                                                        AGCCAGGTGGAAATCCTACAGCGCGTCATCGACTACATTCTCGACCTG240                           SerGlnValGluIleLeuGlnArgValIleAspTyrIleLeuAspLeu                              65707580                                                                      CAGGTAGTCCTGGCCGAGCCAGCCCCTGGACCCCCTGATGGCCCCCAC288                           GlnValValLeuAlaGluProAlaProGlyProProAspGlyProHis                              859095                                                                        CTTCCCATCCAGACAGCCGAGCTCGCTCCGGAACTTGTCATCTCCAAC336                           LeuProIleGlnThrAlaGluLeuAlaProGluLeuValIleSerAsn                              100105110                                                                     GACAAAAGGAGCTTTTGCCACTGA360                                                   AspLysArgSerPheCysHis                                                         115                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysAlaLeuSerProValArgGlyCysTyrGluAlaValCysCys                              151015                                                                        LeuSerGluArgSerLeuAlaIleAlaArgGlyArgGlyLysGlyPro                              202530                                                                        AlaAlaGluGluProLeuSerLeuLeuAspAspMetAsnHisCysTyr                              354045                                                                        SerArgLeuArgGluLeuValProGlyValProArgGlyThrGlnLeu                              505560                                                                        SerGlnValGluIleLeuGlnArgValIleAspTyrIleLeuAspLeu                              65707580                                                                      GlnValValLeuAlaGluProAlaProGlyProProAspGlyProHis                              859095                                                                        LeuProIleGlnThrAlaGluLeuAlaProGluLeuValIleSerAsn                              100105110                                                                     AspLysArgSerPheCysHis                                                         115                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 446 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTTATGACCTCGGAGGAGCTGTGGGCTCGAACCAGTGTTGGGCTAAAGGCGACTGGCAGG60                GGGCAGGGAAGCTCAAAGATCTGGGGTGCTGCCAGGAAAAAGCAAATTCTGAAAGTTAAT120               GGTTTTGAGTGATTTTTAAATCCTTGCTGCCGGAGAGACCCACCTCTCCCCGGTATCAGC180               ACTTCCTCATTCTTTGTATCCACGGCTCCGCGGTCTTCGGCGTCAGACCAGCCGGAGGAA240               GCCTGTTTGCAATTTAAGCGGGCTGTGTACACCCAGGGCCGACGGGGGCGGGGCCGAGGG300               CGGGCCATTTTGAATAAAGAGGCGTGCCTTCCAGGCAGGCTCTATAAGTGACCGCCGCGG360               GCACGTGCGCCGTGCAGGTCACTGTAGCGGGACTTCTTTTGGTTTTCTTTCTCTTTGGGG420               CACCTCTGGACTCACTCCCCAGCATG446                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligodeoxyribonucleotide                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGGGGAGTGAGTCCAGAG18                                                          __________________________________________________________________________

We claim:
 1. Isolated human DNA containing the nucleotide sequence givenin SEQ ID NO: 3, which codes for a Helix-Loop-Helix protein with thedesignation HEIR-1, wherein said DNA naturally falls within theneuroblastoma consensus deletion region 1p36g12.
 2. DNA according toclaim 1, characterised in that it is genomic.
 3. Isolated human genomicDNA characterised in that it contains the sequence shown in SEQ ID NO: 5from position 1 to position
 443. 4. cDNA derived from a transcript ofDNA containing the sequence shown in SEQ ID NO: 3 coding for HEIR-1, ora fragment thereof of at least about 200 bp.
 5. Recombinant DNAcontaining the sequence shown in SEQ ID NO: 3 coding for HEIR-1,functionally connected to expression control sequences, for expressionin a prokaryotic or eukaryotic host cell.
 6. A prokaryotic or eukaryotichost cell, transformed with recombinant DNA according to claim
 5. 7.Isolated HEIR-1 protein of the sequence shown in SEQ ID NO: 4,obtainable by expression of the cDNA defined in claim 5.